1. Field of the Invention
This invention is directed to the discovery that both the nephrotoxic effect of in-progress nephrotoxic therapy on an individual as well as the potential for nephrotoxic effect of contemplated therapy on a given individual can be evaluated as a function of urine kallikrein content.
In the first instance, the urine kallikrein level of a patient receiving such therapy is monitored and compared to the patient's baseline (pretherapy) urine kallikrein level. In the second instance, low baseline urine kallikrein is predictive of patients who are at greater risk for developing nephrotoxic reaction to therapeutic compositions, such as those comprising cyclosporine.
2. Description of the Background Art
Cyclosporine is a cyclic, nonwater-soluble, highly nonpolar molecule composed of eleven amino-acids. The compound is a promising immunosuppressive agent which is derived from soil fungus (Calne et al., Transplant Proc. 13:349-358, (1981); Ferguson et al., Surgery, 92:175-182, (1982); Starzl et al., Gynecol. Obstet., 151:17-26, (1980)). The drug is now widely used for prolonging the function of various transplanted organs. Its immunosuppressive effects selectively inhibit T-cell function, allowing survival of allografts without myelosuppression, i.e. heart transplants, Myers et al., N. Eng. J. Med. 311:699 (1984).
In addition to its use in allograft recipients, recent clinical trials have been or are being undertaken to examine the efficacy of cyclosporine in the treatment of a wide variety of autoimmune diseases, including polymyositis, systemic lupus erythematosus, rheumatoid arthritis, and early insulin dependent diabetes (see relevant chapters in: Cyclosporine in Autoimmune Diseases, ed. Schindler, R., Springer-Verlag, Berlin (1985), particularly von Graffenreid, B. et al., pp. 59-73).
Cyclosporine is a lipophilic molecule with a molecular weight of 1202 daltons. When the drug is dissolved in olive oil or a special solution prepared by the manufacturer, bioavailability and absorption are maximized. The drug readily binds to plasma proteins and has a terminal half-time of 24 hours. It is highly metabolized in the liver, with biliary excretion being the major route of elimination (Beveridge, T., Cyclosporine A:, Proceedings of the International Symposium, Cambridge, White, D. J., ed., pages 35-44 (1982)). In addition to its immunosuppressive characteristics, the drug also has interesting anti-schistosome and anti-malarial activities (Kolata, Science (Washington, D.C.) 221:40-42, (1983); Sanches et al., First Int'l. Montreux Conf. on Biol. Rhythms and Medications, Montreux, Switzerland, Mar. 26-30, 1984. Pergamon Press, Oxford, (in press).
In spite of its great promise as an immunosuppressive, however, its use is somewhat limited, both by its association with infection and also because of hepatic and renal toxicities (Ryffel, OL 27-400: "Summary of Toxicity Data," Sandoz, Basel, Switzerland, (1981)).
Clinical use of cyclosporine is associated with reversible, dose-related increases in blood urea nitrogen (BUN) and serum creatinine levels and depression of creatinine clearance. Some nephrotoxicity is reported to occur in almost 80% of renal transplant patients using cyclosporine (Kahan, B. D., Dial. Transplant., 12:620-30 (1983)). Often the urea nitrogen level is disproportionately increased relative to the serum creatinine level. However, no characteristic changes in the urinary sediment distinguish cyclosporine nephrotoxicity from renal allograft rejection. Thus, the differential diagnosis of rejection in nephrotoxicity largely depends on the presence of associated signs of rejection, such as fever and graft tenderness. Diagnostic difficulties are increased in the early post-transplantation period, when recipients of cadaver allografts are frequently oliguric and dialysis-dependent due to acute tubular necrosis. Renal function in such patients improves with decreasing doses of cyclosporine. (Bennett, W. M. et al., Ann. Int., 99:851-854 (1983)).
In addition, many substances with nephrotoxic potential are present in the environment, particularly in the work place. Lead, mercury, cadmium, and a variety of volatile organic compounds, particularly the halogenated aliphatic hydrocarbons, can produce acute renal disease. The assessment of renal injury based upon serum creatinine or blood urea nitrogen levels is insensitive, these tests demonstrating abnormal findings only when major impairment of renal excretory function has developed. Other measures of renal function, such as creatinine clearance, paraaminohippuric acid clearance, urinary concentrating ability, proteinuria, or glycosuria are, perhaps, somewhat more sensitive, but are technically not satisfactory for use as screening tests, Berndt, W. O., Toxicology of the Kidneys, Hook, J. B., ed., New York: Raven Press, pp. 1-29 (1981); Balazs, T. et al., Toxicol. Appl. Pharmacol., 5:661-674 (1963).
It is known that urinary enzyme analysis provides an extremely sensitive indicator of renal injury. Urinary enzyme levels have been shown to be elevated in a wide spectrum of renal diseases including glomerular and interstitial as well as tubular disease, often prior to the onset of renal failure or even before any abnormality in excretory function is detectable, Price, R. G., Toxicology, 23:99-134 (1982); Maruhn, D., Curr. Probl. Clin. Biochem., 9:135-149 (1979); Piperno, E., supra, pp. 31-55; Sherman, R. L. et al., Arch. Intern. Med., 143:1183-1185 (1983). N-acetyl-beta-D-glucosaminidase, a lysosomal enzyme present in renal tubular cells, has been shown to be an extremely sensitive indicator of early renal injury Well, J. M., Br. Med. J., 3:408-411 (1975); Price, R. G., Curr. Prob. Clin. Biochem., 9:150-163 (1979); Hultberg, B. et al., Clin. Nephrol., 15:33-38 (1981); Kunin, C. M. et al., Pediatrics, 62:751-760 (1978); Viganor, A. et al., Biochem. Med., 25:26-33 (1981); Price, R. G. et al., Clin. Chim. Acta., 27:65-72 (1970); Lockwood, T. D. et al., Toxicol. Appl. Pharmacol., 49:323-336 (1979). Myer, B. R. et al., (Am. J. Med., 76:989-990 (1984)) investigated workers exposed to mercury, lead, or volatile organic compounds and observed elevated urinary acetyl glucosaminidase excretion in these workers. They have proposed this high level of acetyl glucosaminidase as an indication of renal injury.
Kallikrein is an endopeptidase that generates vasoactive polypeptides from kininogens, plasma alpha.sub.2 -globulin substrates (Werle, E. et al., Substanz. Biochem. Z., 289:217 (1937)). It is secreted as an inactive precursor which can be activated in vitro by trypsin (Spragg, Adv. Exp. Med. Biol., 156A:393 (1983)) or thermolysin (Noda et al., Kidney Int., 27:630-635 (1985)). Human urinary kallikrein (urokallikrein or HUK), when purified on the basis of its kinin-generating activity by conventional chromatographic techniques, has been shown to contain a non-kinin-generating alkaline TAMe esterase which can be separated from urokallikrein by broad range isoelectric focusing or by alkaline polyacrylamide disc gel electrophoreses (ole-MoiYoi, O. et al., Biochem. Pharmacol. 26:1893 (1977)). Electrophoretic, antigenic, and immunohistochemical studies have established that urokallikrein and the TAMe esterase represent two distinct renal enzymes (Pinkus, G. S. et al., J. Histochem. Cytochem., Vol. 29, no. 1, 38-44 (1981)). Studies of urokallikrein excretion in physiologic and pathologic circumstances are difficult to interpret utilizing esterolytic assays because of the presence of such nonurokallikrein esterases which cleave the same synthetic substrates (ole-MoiYoi, O. et al., Biochem. Pharmacol. 26:1893 (1977); Levinsky, N. G. et al., Biochem. Pharmacol., 28:2491 (1979)). The use of a tri-peptide p-nitroanilide substrate to measure kallikrein levels is also not specific, since damaged kidneys may filter or secrete non-kallikrein enzymes which cleave this substrate (Koolen et al., Transplantation, 37:471-474 (1984)).
The observation that trypsin treatment of crude urine or a partially purified fraction of urine increases both the esterase (Pisano, J. J. et al., Contrib. Nephrol., 12:116 (1978)) and kininogenase (Spragg, J., Adv. Exp. Med. Biol., 156A:393-398 (1983)) activities of the sample render invalid the use of functional assays of untreated urine samples to determine total kallikrein synthesis and excretion by the kidney.
Silver, M. R. et al., J. Immun., Vol. 124, No. 4, 1551-1555 (1980) developed a radioimmunoassay specific for the active form of urokallikrein utilizing a monospecific antibody that neutralizes the enzymatic activities of urokallikrein and a radioligand purified so as to maintain the active site. Active kallikrein was measured in urine in the absence of trypsin treatment, and total kallikrein was measured after trypsin treatment. Utilizing these radioimmunoassays, the rate of tissue kallikrein excretion into the urine was examined in renal transplant recipients, in post uninephrectomy kidney donors, and in a normal control population (Spragg., J. et al., Kidney International, Vol. 28, 75-81 (1985)). It was observed and reported that kallikrein excretion in uninephrectomized donors and in kidney recipients remained significantly lower than in normal controls. Spragg et al. hypothesized that the reduced kallikrein excretion in postuninephrectomized kidney donors and the even lower excretion in renal allograft recipients suggest that renal kallikrein excretion reflects functional distal tubular mass. Since some of the patients in the Spragg et al. study had received immunosuppressive therapy with cyclosporine, a drug known to have both functional and morphological tubulotoxic effects, it could not be determined whether or not the diminished kallikrein excretion rates in the allograft recipient groups reflected rejection, altered function in a denervated kidney, cyclosporine toxicity or a combination of these factors.
Thus, prior to the present invention, a need had continued to exist for a non-invasive means for evaluating cyclosporine-related nephrotoxicity and a means for predicting potential high-risk cyclosporine therapy patients. In addition, a need has continued to exist for an non-invasive means for evaluating the nephrotoxic effect of other potentially nephrotoxic agents. A need had also continued to exist for a means for distinguishing between nephrotoxicity and allograft rejection in patients being treated with cyclosporine.